PROJECT SUMMARY The inhibitory neurotransmitter GABA produces slow and prolonged inhibition in the brain through activation of metabotropic GABAB receptors (GABABR). Defects in GABABR signaling have been implicated in various neurological and mood disorders including spasticity, epilepsy, addiction and anxiety. GABABR is a member of the class C G protein-coupled receptor (GPCR) family, which typically functions as a dimer and possesses large extracellular domains. Fundamental questions remain concerning the molecular mechanisms underlying activation and modulation of these class C receptors. The GABABR is a heterodimer of GABAB1 and GABAB2 subunits and is modulated by the potassium channel tetramerization domain-containing (KCTD) protein auxiliary subunits. In this proposal, we will develop structural models for full-length GABABR, its auxiliary subunits and their complexes with G- protein. Building on our structural models for both the extracellular and intracellular components of human GABABR, we propose to determine the structural diversity of GABABR auxiliary subunits KCTDs, and elucidate the interactions between KCTDs and G proteins (Aim 1), describe the molecular association between GABABR and KCTD, and determine its impact on GABABR signaling (Aim 2), and solve the structures of full-length GABABR in multiple functional states, characterizing the allosteric interaction between the GABABR and G proteins (Aim 3). We will use an innovative strategy of combining structural and functional analyses, including cryo-electron microscopy (EM) and nanodiscs. Together, these studies will advance our understanding of the molecular basis of GABA action in the brain, leading to the development of novel therapeutics for treating neurological diseases.